WO2005005648A1 - Nouveau procede de production d'acide carboxylique optiquement actif - Google Patents

Nouveau procede de production d'acide carboxylique optiquement actif Download PDF

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WO2005005648A1
WO2005005648A1 PCT/JP2004/009932 JP2004009932W WO2005005648A1 WO 2005005648 A1 WO2005005648 A1 WO 2005005648A1 JP 2004009932 W JP2004009932 W JP 2004009932W WO 2005005648 A1 WO2005005648 A1 WO 2005005648A1
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general formula
represented
acid derivative
cell
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Hiroshi Kawabata
Makoto Ueda
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Mitsubishi Pharma Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

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  • the present invention relates to a carbon-carbon double bond of a 4,18-mouth crotonic acid derivative using enoate reductase, a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell.
  • an optically active 4-halobutyric acid derivative which is a compound useful as an intermediate material for pharmaceuticals, agricultural chemicals and the like.
  • the present invention also provides (R) -N- (4,4,4_trifluoro-2_methinolebutyl) _3_ [2-methoxy_4_ (o_tolylsulfonylcarbamoy) utilizing the method for producing the 4_halobutyric acid derivative. Nore) benzyl] _1-methinoleindol-5-carboxamide.
  • Optically active 4-halobutyric acid derivatives are industrially useful compounds as intermediate raw materials for pharmaceuticals, agricultural chemicals and the like.
  • (R) —4,4,4_trifluoromethyl-2-methylbutyric acid (2R—methyl-4,4,4—trifluorobutanoic acid) is useful as a leukotriene antagonist (R) —4,4 It is known to be an intermediate for the synthesis of 4-methyl-2-butyrylamine, 4-trifluorene (EP-A-489548).
  • Enoate reductase is an enzyme that catalyzes the reaction of reducing the carbon-carbon double bond of enoate (Henoate), and its presence has been reported in microorganisms such as Clostridia (Journal). of Biological Chemistry, vol. 276, No. 8,
  • the present invention also relates to (R) -N- (4,4,4-trifluoro-2-methylbutyl) _3_ [2-methoxy-14- (o-tolylsulfonylcarbamoinole) benzyl] -11-methylindole —To provide a new method for producing carboxamide.
  • the present inventors have conducted intensive studies on a method for producing an optically active 4-halobutyric acid derivative in order to solve the above-mentioned problems.
  • the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 was determined.
  • a transformed cell into which a gene encoding the protein has been introduced is prepared, and the cell, a preparation of the cell, or a culture solution obtained by culturing the cell is used as a starting material for the production of 418-croton.
  • the present invention is as follows.
  • (E) a 4-halocrotonic acid derivative represented by the general formula (I), which has a base sequence that hybridizes under stringent conditions with the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary sequence thereof, DNA encoding a protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative represented by the formula ( ⁇ ).
  • nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 one or several nucleotides are substituted, deleted or added, and comprise a complementary nucleotide sequence and represented by the general formula (I)
  • R is an S methyl group
  • R represents a halogen atom, a nitro group, a hydroxy group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted X is a halogen atom;
  • a and A are a hydrogen atom or a halo group; an aryl group or an optionally substituted alkoxy group;
  • examples of the alkyl group include a methynole group, an ethyl group, an n-propyl group, an isopropyline group, a cyclopropyl group, an n_butyl group, an isobutyl group, a tertiary butyl group, an n-pentyl group, Examples include straight-chain, branched-chain and cyclic alkyl groups having 118 carbon atoms such as isopentyl group, neopentyl group, tertiary pentyl group, isoamyl group, n-hexyl group and the like.
  • a methyl group or an ethyl group is particularly preferable, and an alkyl group having 14 to 14 carbon atoms is particularly preferable.
  • aryl groups include phenyl, mesityl, and naphthyl. I can get lost.
  • the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a tertiary butoxy group, and the like. Among them, an alkoxy group having 14 to 14 carbon atoms is preferable.
  • the alkyl group, aryl group and alkoxy group may be substituted.
  • the substituent is not particularly limited as long as it does not adversely affect the asymmetric reduction reaction, but specifically, an alkyl group, an aryl group, an alkoxy group, a halogen group, a cyano group, an amino group, Examples include a nitro group and a hydroxy group. Therefore, in the general formulas (I) and ( ⁇ ), specific examples of the substituted alkyl group include a benzyl group, a phenethyl group, a trifluoromethyl group, a cyanomethyl group, an aminomethyl group, a hydroxymethyl group, a nitromethyl group, and a methoxymethyl group.
  • a xymethyl group and the like include a chlorophenyl group, an aminophenyl group, a hydroxyphenyl group, a nitrophenyl group, and a methoxyphenyl group.
  • Specific examples of the substituted alkoxy group include a benzyloxy group, a phenoxy group, and a trifluoromethoxy group.
  • X represents a halogen atom
  • a and A represent a hydrogen atom or a halogen atom.
  • R in the above general formulas (I) and (II) is preferably an alkyl group having 14 to 14 carbon atoms, a benzyl group or a phenyl group, and more preferably an alkyl group having 14 to 14 carbon atoms. And particularly preferably a methyl group.
  • the compound represented by the general formula (I) has a molecular weight of 1,000 or less, preferably 500 or less, more preferably 300 or less.
  • the compound represented by the general formula (III) 4,4,4_Trifluorotiglic acid and the like.
  • the compound represented by the general formula (I) is, for example, an aldehyde represented by the following general formula (VIII) (X, A
  • the compound represented by the general formula ( ⁇ ) has a molecular weight of 1000 or less, preferably 500 or less, more preferably 300 or less.
  • Enoate reductase generally refers to an enzyme that catalyzes the reduction reaction of the carbon'carbon double bond of the enoate (Henoate) (Studies in Natural Products Chemistry, vol. 20, p817, 1998).
  • the optical purity of the generated 4-halobutyric acid derivative is preferably at least 60% ee, more preferably at least 90% ee, and at least 98% ee. It is particularly preferred that the ee is 99.5% ee.
  • Such an activity can be measured by measuring the initial rate of decrease of NADH in a reaction system containing a 4-nitrocrotonic acid derivative as a substrate and further containing NADH as a coenzyme.
  • the enoate reductase that can be used in the production method of the present invention is not particularly limited as long as it has the above activity, and examples thereof include those having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. These are enoate reductases derived from Moorella thermoautotrophica and Clostridium acetobutylicum, respectively. Further, in the present invention, homologs of these homologs having the above-mentioned enzyme activity may be used. Homologs include, for example, those having an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 within a range that does not impair the activity. be able to.
  • the term “several” specifically means 20 or less, preferably 10 or less, and more preferably 5 or less.
  • the homolog has 35% or more, preferably 50% or more, more preferably 80%, particularly preferably 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. It may be protein.
  • the homology search of the above-mentioned protein can be performed using, for example, a program such as FASTA or BLAST for GenBank or DNA Databank of JAPAN (DDBJ).
  • a homology search was performed using the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 with the BLAST program against GenBank, and as a result, the sequence of SEQ ID NO: 2 was 2-enoate reductase derived from Clostridium tyrobutyricum (Accession No. CAA71086) and 59% showed homology, eye ti ⁇ 1 of SEQ ID NO: 4 J is Clostridium Tyrobutyricum from 2- enoate reductase (Accession No.
  • the enoate reductase used in the production method of the present invention may be any microorganism having enoate reductase activity using a probe prepared based on the nucleotide sequence of a gene encoding a part or all of enoate reductase.
  • a method for obtaining enoate reductase from bacterial cells for example, the method described in Eur. J. Biochem. Vol. 97, pl03 (1979) can be referred to.
  • Clostridium bacteria include, for example, Clostridium acetobutylicum ATCC824 strain, which has enoate reductase that can be suitably used in the present invention.
  • Clostridium acetobutylicum ATCC824 strain which has enoate reductase that can be suitably used in the present invention.
  • a bacterium belonging to the genus Moorella for example, Moorella thermoautotrophica
  • the strain DSM1974 has enoate reductase that can be suitably used in the present invention.
  • the former is an ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (ATCC (American Type Culture Collection) online catalog (AT
  • the 4_nodrocrotonic acid derivative may be allowed to react with a purified enzyme of enoate reductase, but a cell containing enoate reductase, a preparation of the cell, or the cell is used.
  • the culture solution obtained by culturing may be reacted with a 4,18-mouth crotonic acid derivative to produce an optically active 4,18-mouth butyric acid derivative.
  • the cell containing enoate reductase is preferably a cell transformed with DNA encoding enoate reductase.
  • the DNA encoding enoate reductase includes a DNA encoding enoate reductase having the amino acid sequence of SEQ ID NO: 2 or 4.
  • a 4-halocrotonic acid derivative having an amino acid sequence IJ having 50% or more homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and represented by the general formula (I) is represented by the general formula ( ⁇ ) )) May be a DNA encoding a protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative.
  • a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 can be mentioned.
  • Such DNA can be obtained by PCR using a primer designed based on the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Furthermore, in the production method of the present invention, a DNA homolog of a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having an enoate reductase activity may be used.
  • a homolog is defined as a protein having an enoate reductase activity, in which one or several bases are deleted, substituted, or added to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3.
  • severe specifically refers to 60 or less, preferably 30 or less, and more preferably 10 or less. It is.
  • the homolog may be a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof under stringent conditions and encodes a protein having an enoate reductase activity.
  • DNA that hybridizes under stringent conditions refers to a method using a probe DNA and a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method under stringent conditions.
  • stringent conditions refers to DNA obtained by performing the hybridization method or the like.
  • a 0.1 X 2 SSC solution After performing hybridization at 65 ° C in the presence of 0.7-1.0 M sodium chloride using a filter on which the DNA or a fragment of the DNA has been immobilized, a 0.1 X 2 SSC solution (The composition of 1 X SSC is 150 mM sodium chloride, 15 mM sodium citrate). Conditions for washing the filter under C conditions can be mentioned.
  • DNA encoding enoate reductase can be isolated, for example, by the following method.
  • enoate reductase is purified from microbial cells or the like by the above-mentioned method or the like, and then the N-terminal amino acid sequence is analyzed.
  • N-terminal amino acid sequence analysis is performed by cleaving the purified protein with enzymes such as lysyl endopeptidase and V8 protease, purifying the peptide fragment by reverse-phase liquid chromatography, etc., and then analyzing the amino acid sequence with a protein sequencer.
  • the DNA encoding the enoate reductase is obtained.
  • the above DNA fragment is probed from a library or cDNA library obtained by introducing a restriction enzyme digest of chromosomal DNA of an enoate reductase-producing microorganism strain into a phage, a plasmid, etc., and transforming E. coli.
  • colony hybridization, plaque hybridization, and the like DNA encoding enoate reductase can be obtained.
  • DNA encoding enoate reductase can be obtained by performing invese PCR (Genetics vol. 120, p621-623 (1988)) as type ⁇ .
  • the DNA encoding enoate reductase can also be obtained by chemically synthesizing DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • DNA encoding enoate reductase was prepared from any microorganism having enoate reductase activity using a DNA having all or a part of the base sequence of SEQ ID NO: 1 or 3 as a probe.
  • the DNA is subjected to hybridization under stringent engineering conditions by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, and hybridized. It can also be obtained by obtaining DNA.
  • the term “part” refers to a DNA having a length sufficient to be used as a probe, specifically, 15 bp or more, preferably 50 bp or more, more preferably 100 bp or more.
  • the enoate reductase expression vector is provided by inserting the DNA encoding enoate reductase isolated as described above into a known expression vector so that it can be expressed. By culturing cells transformed with this expression vector, enoate reductase can be obtained from the cells. Transformed cells can also be obtained by incorporating DNA encoding enoate reductase into a chromosomal DNA of a known host cell so that it can be expressed.
  • an expression vector constructed by incorporating DNA encoding enoate reductase into a plasmid vector or a phage vector stably existing in a microorganism is used.
  • the DNA encoding enoate reductase must be introduced directly into the host genome and the genetic information must be transcribed and translated.
  • the DNA encoding the enoate reductase does not contain a promoter that can be expressed in the host microorganism, an appropriate promoter is placed 5 'upstream of the DNA strand encoding the enoate reductase. Must be incorporated. Further, it is preferable to incorporate a terminator downstream of the 3 'side.
  • the promoter and terminator are not particularly limited as long as they are known to function in a microorganism used as a host, provided that they are promoters and terminators. Regarding promoters and terminators, for example, "Basic Lectures on Microbiology 8Genetic Engineering 'Kyoritsu Shuppan'," especially for yeast, Adv. Biochem. Eng. Vol. 43, P75-102 (1990), Yeast vol. 8, It is described in detail in p423_488 (1992).
  • the host microorganism to be transformed for expressing the enoate reductase of the present invention is not particularly limited as long as the host itself does not adversely affect the present reaction.
  • the following microorganisms can be mentioned.
  • Actinomycetes having an established host vector system belonging to the genera Rhodococcus, Streptomyces, and the like.
  • the host is preferably a genus Escherichia, a genus Bacillus, a genus Brevibacterium, or a corynebacterium (
  • Corynebacterium and particularly preferably, Escherichia and Corynebacterium j.
  • plasmid vectors include pBR and pUC-type plasmids, and promoters include lac (i3-galatatosidase) and trp (tryptophan operon). , Tac, trc (fusion of lac and trp), ⁇ phage PL, PR and the like.
  • the terminator include a terminator derived from trpA, phage, and rrnB ribosomal RNA.
  • vectors include pUBlO-based plasmid and pC194-based plasmid. And chromosomes.
  • promoters and terminators of enzyme genes such as alkaline protease, neutral protease and ⁇ -amylase can be used.
  • vectors include Pseudomonas' putida (
  • examples of the vector include a plasmid vector such as pAJ43 (Gene vol. 39, p281 (1985)).
  • pAJ43 Gene vol. 39, p281 (1985)
  • Various promoters and terminators used in Escherichia coli can be used as the promoter and terminator.
  • Corynebacterium particularly Corynebacterium 'daltamicum (Corynebacterium
  • examples of the vector include a plasmid vector such as pCS11 (Japanese Patent Publication No. 57-183799, pCB101 (Mol. Gen. Genet, vol. 196, pl 75 (1984))).
  • Saccharomyces especially Saccharomyces cerevisiae
  • examples of the vector include YRp, YEp, YCp, and Yip plasmids.
  • promoters and terminators of various enzyme genes such as alcohol dehydrogenase, dalyselanoledehydrido 3-phosphate dehydrogenase, acid phosphatase, / 3_galactosidase, phosphodarycelate kinase, and enolase can be used.
  • examples of the vector include a plasmid vector derived from Schizosaccharomyces bomb described in Mol. Cell. Biol. Vol. 6, p80 (1986). .
  • pAUR224 is commercially available from Takara Shuzo and can be easily used.
  • Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is available. Yes, and a promoter derived from extracellular protease ⁇ amylase is available (Trends in
  • host vector systems corresponding to various microorganisms have been established, and these can be used as appropriate.
  • various host systems have been established in plants and animals, especially in animals such as insects using silkworms (Nature vol. 315, p592-594 (1985)), rapeseed, A system for expressing a large amount of a heterologous protein in plants such as corn and potato and a system using a cell-free protein synthesis system such as a cell-free extract of Escherichia coli or wheat germ have been established and can be suitably used.
  • a cell transformed with a DNA encoding enoate reductase may be allowed to act on a 4,18-mouth crotonic acid derivative as a reaction substrate.
  • the transformed cells may be allowed to act as they are in the reaction solution.
  • a preparation of the transformed cells for example, the transformed cells may be treated with an organic solvent such as acetone, dimethyl sulfoxide (DMS ⁇ ), toluene, or a surfactant.
  • a culture solution obtained by culturing the cells that is, a culture solution containing the cells may be directly used.
  • coenzyme NAD + or NADH it is preferable to add coenzyme NAD + or NADH to the reaction solution.
  • concentration of the additive is 0.001 mM, preferably 100 mM.
  • regenerate NAD + generated from NADH into NADH it is preferable to regenerate NAD + generated from NADH into NADH as a preferable regeneration method for improving production efficiency.
  • Microorganisms or their preparations capable of producing NADH from NAD + , or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase A method of adding an enzyme (regenerating enzyme) that can be used to regenerate NADH, such as elemental or organic acid dehydrogenase (malate dehydrogenase, etc.), or
  • microorganisms containing the above-mentioned regenerated enzymes those obtained by treating the microorganism cells with acetone, those subjected to freeze-drying, those physically or enzymatically crushed, and the like.
  • a cell preparation, a product obtained by extracting the enzyme fraction as a crude product or a purified product, or a product obtained by immobilizing these on a carrier represented by polyacrylamide gel, carrageenan gel, etc. may be used.
  • a commercially available regenerating enzyme may be used.
  • the amount of the regenerating enzyme used is, specifically, about 0.01 to 100 times, preferably about 0.5 to 20 times the enzyme activity as compared with the enoate reductase of the present invention.
  • Compounds that serve as substrates for the above-mentioned regenerating enzymes for example, glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. Force required to be added
  • the amount of addition is 0.1 to 20 times molar equivalent, preferably 115 to 5 times molar equivalent, to the 4-halocrotonic acid derivative as a reaction raw material.
  • the production method of the present invention contains, for example, a reaction substrate, the transformed cell of the present invention and / or the transformed cell preparation, various coenzymes added as necessary, and a regeneration system thereof.
  • the reaction can be carried out in an aqueous medium or in a mixture of the aqueous medium and an organic solvent.
  • the compound represented by the general formula (I) serving as a reaction substrate in the production method of the present invention generally has a substrate concentration of 0.01% 90% w / v, preferably 0.1% to 30% w / v. It can be used within the range.
  • the reaction substrate may be added all at once at the beginning of the reaction, but from the viewpoint of reducing the effects of enzyme substrate inhibition and improving the accumulated concentration of the product, continuous or It is desirable to add intermittently.
  • the aqueous medium includes water or a buffer
  • the organic solvent includes a reaction substrate such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and dimethyl sulfoxide.
  • a reaction substrate such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and dimethyl sulfoxide.
  • the one with high solubility can be used.
  • the method of the present invention can be carried out, for example, at a reaction temperature of 4 to 60 ° C, preferably 10 to 45 ° C, and at pH 3 to 11, preferably pH 5 to 8. It is also possible to carry out the reaction using a membrane reactor or the like. It is also effective to remove sodium by adding sodium sulfite to the reaction solution or sealing the reaction solution with nitrogen or argon gas to prevent inactivation of enoate reductase by oxygen. It is.
  • the optically active 4-halobutyric acid derivative produced by the method of the present invention is obtained by separating the bacterial cells in the reaction solution by centrifugation or membrane treatment after completion of the reaction, and then removing the organic compound such as ethyl acetate or toluene. Purification can be performed by appropriately combining extraction with a solvent, distillation, column chromatography, crystallization, and the like.
  • Step (a) can be performed in the same manner as described above.
  • a usual method for converting a carboxylic acid to an amine can be used.
  • a method of reacting with ammonia to amidate and then reducing (reaction formula (IX) below), reacting with a pendinoleamine or the like or an ammonia equivalent (eg, HN (SiMe), etc.), and then reducing the reactant And more contact
  • a deprotection method such as hydrogenolysis (reaction formula (X)) can be used.
  • step (c) (R) _4,4,4-monotrifluoro-2-methylbutynoleamine of formula (V) obtained in step (b) is reacted with a compound of formula (VI) to give a compound of formula (VI) (VII) (R) -N- (4,4,4-Trifluoro 2-methylbutyl) _3_ [2-Methoxy-1- (o-tolylsulfonylcarbamoyl) benzyl] -1-1-methylindole-5_carboxamide Get.
  • This step can be carried out in the same manner as in the usual reaction between amine and carboxylic acid. Specifically, for example, the method is performed in the same manner as disclosed in European Patent Application Publication No.
  • the compound of the general formula (VI) used in this step may be a compound obtained by the above-mentioned production method.
  • 4_ (5-methoxycarbonyl) 1-Methylindole-3-ylmethyl) _A compound obtained by reacting 3-methoxybenzoic acid (general formula XI) with 2-methylbenzenesulfonamide (general formula ⁇ ) and hydrolyzing the resulting compound can be used.
  • 3-methoxybenzoic acid generally formula XI
  • 2-methylbenzenesulfonamide generally formula ⁇
  • the above reaction solution was purified using MinElute PCR Purification kit (manufactured by Qiagen). Purification The DNA fragment thus obtained was digested with restriction enzymes EcoRI and Xbal, subjected to agarose gel electrophoresis, excised from the band of interest, purified and recovered using a Qiagen Gel Extraction kit (manufactured by Qiagen). The obtained DNA fragment was ligated to pUCl18 digested with EcoRI and Xbal using Ligation high (manufactured by Toyobo Co., Ltd.), and Escherichia coli JM109 strain was transformed by a heat shock method.
  • the transformed cells were grown on an LB agar medium containing ampicillin (50 ag / mL), and colony direct PCR was performed on the obtained colonies to confirm the size of the imported fragment.
  • the transformed cells containing the DNA fragment of interest are cultured in an LB medium containing 50 ⁇ g / mL ampicillin, and the plasmid is purified using the QIAPrepSpin Mini Prep kit (Qiagen). , PUCMtERl.
  • the base sequence of the DNA inserted into the plasmid was analyzed by the dye terminator method, the inserted DNA fragment was identical to the nucleotide sequence of SEQ ID NO: 1.
  • DNA sequence encoding 2-enoate reductase (Accession No. AAK81302, SEQ ID NO: 4) derived from Clostridium acetobutylicum
  • primers described in SEQ ID NOs: 7 and 8 were synthesized. These primers were used in an amount of 15 pmol each, dNTP each 10 nmol, genomic DNA of Clostridium acetobutylicum ATCC824 25 ng, 10X buffer for KOD-plus— (Toyobo) 5 ⁇ L, KOD-plus-1 unit ( with 50's reaction solution containing Toyobo Co., Ltd.), modified (94 ° C, 15 ⁇ ), Aninore (57.G, 30 ⁇ ), I monkey length (68 0 C, 2 min) 30 A PCR reaction was performed using PTC-200 (manufactured by MJ Research) in a cycle. As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, a band considered to be specific was detected.
  • the above reaction solution was purified using MinElute PCR Purification kit (manufactured by Qiagen).
  • the purified DNA fragment was digested with restriction enzymes EcoRI and Xbal, subjected to agarose gel electrophoresis, excised a band of interest, purified using a Qiagen Gel Extraction kit (manufactured by Qiagen), and collected.
  • the obtained DNA fragment was digested with EcoRI and Xbal.
  • ligation was performed using Ligation high (manufactured by Toyobo Co., Ltd.), and Escherichia coli JM109 strain was transformed.
  • the transformed cells were grown on LB agar medium containing ampicillin (50 ⁇ g / mL), and the resulting colonies were subjected to colony direct PCR to confirm the size of the inserted fragment.
  • the transformed cells containing the DNA fragment of interest are cultured in an LB medium containing 50 xg / mL ampicillin, and the plasmid is purified using the QIAPrepSpin Mini Prep kit (Qiagen). pUCCaERl.
  • the nucleotide sequence of the DNA inserted into the plasmid was analyzed by a dye terminator method, the inserted DNA fragment was identical to the nucleotide sequence of SEQ ID NO: 3.
  • the transformed cells obtained in the above (1) and (2) were subjected to Aneropaque's culture in an LB medium containing ampicillin (50 x gZmL) and ImM isopropyl ⁇ -D-thiogalatatopyranoside (IPTG). (Mitsubishi Gas Chemical Company) under anaerobic conditions at 37 ° C for 30 hours. After 5 ml of the obtained cell broth was collected by centrifugation to obtain cells, the enoate reductase activity of the cells was determined using 4,4,4 trifluorotiglic acid as a substrate by the method described below. Sure
  • the substrate 4,4,4 trifluorotiglic acid was synthesized as follows. That is, 50% water-containing methanol (20 mL) and 8 mol / L NaOH (3 mL) were added to 4,4,4_trifluoroethyltignate ethyl ester (3.64 g, 20 mmol), and the obtained mixture was added to about 4 mL. Stir for 2 hours. The solvent was distilled off from the reaction mixture under reduced pressure, the pH was adjusted to 2 or less by adding concentrated hydrochloric acid, methylene chloride extraction was performed, and the obtained organic layer was concentrated under reduced pressure to obtain a transparent oily product as a transparent oil. Trifluorotiglic acid (2.9 g, yield 94%) was obtained.
  • a 200 ⁇ L reaction solution (0.6 g / L NAD + (manufactured by Oriental Yeast), 50 mM potassium phosphate buffer (pH 7.0), 100 mM glucose, 0.2 g ZL glucose dehydrogenase (Amano) was added to the cells.
  • 25 mM 4,44_triphnorelotiglic acid (manufactured by Pharmaceutical Co., Ltd.)
  • the mixture was reacted at 37 ° C. for 20 hours under anaerobic conditions using Anellopak'Kenki (manufactured by Mitsubishi Gas Chemical Company).
  • the reaction was terminated by adding 10 ⁇ L of 6N HC1, and the mixture was extracted with ethyl acetate, and 2-methyl-4,4,4_trifluorobutyric acid was quantified. Determination is ethyl acetate The solution was measured using gas chromatography (GC).
  • GC gas chromatography
  • Carrier He 1.5 ml / min, split 1/50

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Abstract

Selon la présente invention, on prend une énoat-réductase, des cellules en contenant, une préparation comprenant ces cellules, ou une culture obtenue à partir de ces cellules, et on les fait agir sur un dérivé d'acide 4-halocrotonique représenté par la formule générale (I). On obtient ainsi un dérivé dacide 4-halobutyrique représenté par la formule générale (II). Dans ces formules, R est halogène, nitro, hydroxy, alkyle éventuellement substitué, aryle éventuellement substitué, ou alcoxy éventuellement substitué, A1 et A2 repérsentant chacun un hydrogène ou un halogène.
PCT/JP2004/009932 2003-07-11 2004-07-12 Nouveau procede de production d'acide carboxylique optiquement actif WO2005005648A1 (fr)

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WO2017004709A1 (fr) * 2015-07-03 2017-01-12 The Governing Council Of The University Of Toronto Procédé et micro-organisme pour la synthèse d'acide adipique à partir d'acides carboxyliques

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007115819A1 (fr) 2006-04-11 2007-10-18 Hauzer Techno Coating Bv Appareil de traitement sous vide, source de courant de polarisation et procédé d'exploitation d'un appareil de traitement sous vide
WO2017004709A1 (fr) * 2015-07-03 2017-01-12 The Governing Council Of The University Of Toronto Procédé et micro-organisme pour la synthèse d'acide adipique à partir d'acides carboxyliques
US11124778B2 (en) 2015-07-03 2021-09-21 The Governing Council Of The University Of Toronto Process and microorganism for synthesis of adipic acid from carboxylic acids

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